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2-Macroglobulin in Regulation of Estradiol Production by Granulosa Cells and Development of Dominant Follicles
Molecular Reproductive Endocrinology Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824-1225
Address all correspondence and requests for reprints to: J. J. Ireland, Molecular Reproductive Endocrinology Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824-1225. E-mail: ireland{at}msu.edu.
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Abstract |
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2-Macroglobulin (2-M) inhibits proteinases and modulates the actions of growth factors and cytokines. Despite the key roles proteinases, growth factors, and cytokines have in folliculogenesis, the role of 2-M in follicular development is unknown. Our objectives were to: 1) determine whether granulosa cells produce 2-M and have 2-M receptors, 2) examine the effect of 2-M on estradiol production by granulosa cells, 3) establish whether amounts of 2-M and 2-M receptors were altered during dominant nonovulatory follicle development, and 4) examine 2-M’s mechanism of action. The results demonstrated that bovine granulosa cells contain 5.2- and 15-kb mRNAs and 720- and 500-kDa proteins that correspond, respectively, to sizes of mRNAs and proteins for 2-M and the 2-M receptor. Treatment of granulosa cells with 2-M resulted in a specific dose-responsive increase in estradiol production. Cell viability, cell number, and the amount of aromatase in granulosa cells were not altered by 2-M. Treatment of granulosa cells with factors that bind 2-M or its receptor did not mimic 2-M action. Although intrafollicular amounts of 2-M remained unchanged, amounts of 2-M receptor in granulosa cells were strongly inversely associated with concentrations of estradiol in dominant and subordinate follicles. Based on these results, we concluded that 2-M may have autocrine or paracrine roles in granulosa cells potentially important for regulation of estradiol production and development of dominant follicles. |
Introduction |
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2 -MACROGLOBULIN (2-M) is a 720-kDa tetrameric glycoprotein that belongs to a superfamily of proteinase inhibitors and complement components (1). The unique tertiary, cage-like structure of 2-M enables it to inhibit the activity of proteinases (2) and bind and enhance or inhibit the biological activity of a variety of growth factors (1). Native 2-M does not bind receptors (1). However, after native 2-M binds proteinases or is oxidized, it is conformationally transformed (3, 4, 5, 6). Conformational transformation of 2-M exposes its receptor binding domains, thus enabling it to bind membrane receptors (1). The 2-M receptor, also known as the low-density lipoprotein receptor-related protein or CD91 (7), is highly conserved among species (8) and is one of the largest known cell surface receptors (
585 kDa) (8), and it binds numerous ligands including transformed 2-M (1). Once ligands are bound to the 2-M receptor, they are internalized and degraded by lysosomes (9). Thus, interaction of transformed 2-M with its receptor depletes not only 2-M but also the proteinases and growth factors bound to transformed 2-M from blood, tissues, and extravascular spaces. In addition, interaction of transformed 2-M with receptors activates a cAMP-dependent kinase signaling pathway (10), stimulates calcium uptake by cells (11), and activates a tyrosine kinase class of receptors similar to epidermal growth factor or insulin (12, 13). However, once native 2-M binds proteinases or is oxidized and thus transformed, the transformed 2-M loses its capacity to bind and inhibit proteinases but retains its ability to bind growth factors or cytokines (1). Thus, conformational transformation of 2-M by proteinases or oxidation has an important role in regulation of the biological actions of 2-M.
Despite 2-M’s dual role as a growth factor binding protein and proteinase inhibitor, the physiological role of 2-M in ovarian follicular growth, differentiation, and function has to our knowledge never been investigated. 2-Macroglobulin is produced by granulosa and thecal cells in rats (14, 15, 16), and 2-M receptor mRNA is present in granulosa cells of humans and rats (1, 9). In addition, intrafollicular 2-M concentration is 80–90% lower, compared with bovine serum or plasma (17, 18), and averages 0.6, 1.0, and 1.2 mg/ml of follicular fluid for small (1–5 mm), medium (6–13 mm), and large (>15 mm) bovine follicles (18). Taken together, these findings imply that 2-M in bovine (b) follicular fluid (FF) of antral follicles may be produced primarily by follicular cells and that enhanced production of 2-M by granulosa and thecal cells during follicle growth, and interaction of transformed 2-M with its receptor may be important for follicular development. In support of a role of 2-M in follicular growth and function, 2-M binds most of the growth factors, binding proteins, or hormones that either enhance [TGFß (19, 20), TGF (21, 22), activin (20), insulin (23)], or inhibit [follistatin (24, 25), epithelial growth factor (EGF) (26, 27), TGF (22, 27, 28, 29), activin (30), TGFß1 (30), basic fibroblast growth factor (31), inhibin (19, 32, 33)] estradiol production. Thus, intrafollicular 2-M could alter capacity of granulosa cells to produce estradiol in several ways: 1) regulating activity of intrafollicular proteinases, 2) exerting a broad stimulatory or inhibitory effect on the biological action of the aforementioned factors that interact with gonadotropins, 3) interacting with its receptor on granulosa cells to deplete from follicular fluid the proteinases, hormones, or growth factors bound to 2-M, and/or 4) interacting with 2-M receptors on granulosa cells to exert a direct effect on estradiol production. Therefore, we hypothesized that 2-M has an important role in regulation of the intrafollicular capacity of granulosa cells to produce estradiol, which is the key physiological event required for development of dominant follicles during nonovulatory and ovulatory follicular waves in cattle (34, 35), and perhaps in other single-ovulating species with follicular waves including humans (36, 37).
To begin to test this hypothesis in cattle, the objectives of our study were to: 1) determine whether granulosa cells produce 2-M and have 2-M receptors, 2) examine the effect of 2-M on basal capacity of granulosa cells to produce estradiol, 3) establish whether intrafollicular amounts of 2-M and the 2-M receptor in granulosa cells and the biological action of 2-M are altered during development of dominant and subordinate follicles, and 4) examine the mechanism of action of 2-M.
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Materials and Methods |
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2-M (h2-M) cDNA; BACPAC Resources (Oakland, CA), b2-M receptor cDNA; PerkinElmer Inc. (Boston, MA), 32P-dCTP, 32P-dATP, and GeneScreen Plus; Sigma (St. Louis, MO), 19-hydroxyandrostenedione, h2-M, bIgG, bovine trypsin type I, bovine insulin, human lactoferrin, low-density lipoprotein (ß-lipoprotein from human plasma), glutathione, isobutyl-methylxanthine, dibutyryl cAMP, and Percoll; Serologicals Corp. (Kankakee, IL), BSA; Bio-Rad Laboratories, Inc. (Hercules, CA), Triton X-100, DC protein assay kit, broad-range protein standards, and Econo-Pac 10DG desalting columns; Roche Diagnostics Corp. (Indianapolis, IN), b2-M and Complete, miniprotease inhibitor cocktail tablets; Biogenesis, Inc. (Sandown, NH), polyclonal rabbit anti-b2-M; Serotec (Oxford, UK), monoclonal against an epitope in extracellular region of 500-kDa -chain of h2-M receptor or CD91; Dr. N. Groome (Oxford Brookes University, Oxford, UK), monoclonal antibody against a peptide fragment corresponding to amino acids 376–390 of human aromatase; Amersham Pharmacia Biotech (Piscataway, NJ), donkey antirabbit IgG- and sheep antimouse IgG-horseradish peroxidase linked to whole antibodies, enhanced chemiluminescence Western blotting detection reagents and high-molecular-weight calibration kit; Dr. A. F. Parlow (National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA), bFSH (USDA-bFSH-B1, AFP-5700), ovine FSH (NIDDK-oFSH-20, AFP-7028C), bLH (USDA-bLH-B6, AFP-11743-B), and follistatin (rhFS-228, code 84384); Peninsula Laboratories Inc. (San Carlos, CA), IGF-I; R & D Systems (Minneapolis, MN), TGF, TGFß1, EGF, antihuman TGF, and pan-specific anti-TGFß; Bachem (Torrance, CA), osteogenic growth factor; Austral Biologicals (San Ramon, CA), IGF binding protein-4; Dr. M. F. Smith (University of Missouri, Columbia, MO), recombinant ovine tissue inhibitor of metalloproteinases (TIMP)-1; Millipore Corp. (Bedford, MA), Microcon YM-3 centrifugal devices and Immobilon-P; Kodak (Rochester, NY), X-Omat Blue XB-1; Becton Dickinson and Co. (Lincoln Park, NJ), Falcon Primaria plates; Diagnostic Products Corp. (Los Angeles, CA), estradiol double antibody and Coat-A-Count progesterone kits.
Sources and procedures to isolate bFF and granulosa cells from follicles
Unless specified otherwise, granulosa cells were isolated from dominant nonovulatory or subordinate follicles of ovaries obtained at a local abattoir, as previously explained (33, 38). In brief, ovaries between d 2 and 10 of an estrous cycle based on morphology of the corpus luteum (39) were obtained from the abattoir and transported to the laboratory in ice-cold PBS. Days 2–10 of an estrous cycle correspond to the days of development for first-wave dominant nonovulatory follicles (40). The largest or dominant follicle, and in some studies, the dominant and the two largest subordinate follicles per pair of ovaries were isolated from ovarian stroma. The bFF was aspirated, centrifuged to remove cell debris, and stored at –20 C for subsequent determination of concentrations of estradiol and progesterone. In some studies, ratio of concentration of estradiol to progesterone in bFF was used to separate first-wave dominant nonovulatory and subordinate follicles in hindsight into two categories: estrogen active (estradiol > progesterone in bFF), or estrogen inactive (progesterone > estradiol in bFF). Estrogen-active follicles have biochemical characteristics of healthy growing dominant follicles, whereas estrogen-inactive follicles are destined for atresia (41, 42). Granulosa cells isolated from each follicle were washed in Ham’s F12 media and then cultured under serum-free conditions. Elapsed time from collection of ovaries at the abattoir (30 min after death of animal) until initiation of culture was 4–5 h.
Granulosa cells were also isolated from dominant ovulatory and nonovulatory follicles of conscious cows, as previously explained (33). All studies using cattle were sanctioned by the All University Committee on Animal Use and Care at Michigan State University. In our study, granulosa cells were obtained from 14 dominant ovulatory and 12 first-wave dominant nonovulatory follicles from the same 14 conscious, nonlactating, multiparous Holstein cows (4–7 yr of age) using ultrasound-guided needle biopsy. After aspiration and removal of red blood cells with use of a 45% Percoll column, number of granulosa cells recovered per follicle averaged 2.56 x 105 ± 0.67 (± SEM). Because numbers of granulosa cells isolated from individual follicles were highly variable, each study used cells from either an individual follicle or a pool of follicles as follows. For dominant ovulatory follicles (n = 5 studies), granulosa cells were isolated from 14 dominant follicles of 14 cows. Cells from two of the 14 follicles were cultured independently, whereas cells from the remaining 12 follicles were used to form three different pools of cells, which were cultured independently. For dominant nonovulatory follicles (n = 6 studies), granulosa cells were isolated from 12 dominant nonovulatory follicles of the same 14 cows. Cells from these 12 follicles were then used to form six different pools, and each pool was cultured independently. Total elapsed time from granulosa cell aspiration until initiation of cell culture was approximately 2 h. The Coulter Counter Particle Z1 (Beckman Coulter, Fullerton, CA) was used to determine cell number, and Trypan blue exclusion dye was used to estimate cell viability at the beginning and end of culture (33, 38).
RNA extraction
Granulosa cells isolated from dominant and subordinate follicles were placed in 100 µl Dulbecco’s PBS (DPBS, pH 7.2), frozen in liquid nitrogen within 40 min of slaughter of each cow, and stored at –80 C. Total RNA was extracted with use of Trizol per manufacturer’s instructions. Total RNA was then pooled because amounts of mRNA for b2-M and b2-M receptor were too low to detect in individual follicles by Northern blot analysis. Poly(A)+ RNA was isolated from total RNA with use of MessageMaker and FastTrack 2.0 kits.
Preparation of b2-M and b2-M receptor cDNA probes
Plasmid DNA was isolated with use of the Wizard Plus miniprep DNA purification system. Plasmid inserts were isolated by restriction enzyme digestion (b2-M, 542 bp, XhoI and BamHI; b2-M receptor, 3 kb, XbaI and SalI), agarose gel electrophoresis, and purification by a unidirectional electroeluter (IBI, New Haven, CT). The b2-M cDNA corresponding to nucleotides 3398–3884 of the h2-M cDNA (GenBank accession no. M11313) was cloned from bovine corpus luteum into pBluescript II SK. The b2-M receptor cDNA corresponding to an expressed sequence tag (GenBank accession no. AW669980) was cloned into polyclonal cytomegalovirus SPORT 6. Probes for hybridizations were generated from the plasmid inserts by the random primers DNA labeling system with use of 32P-dCTP and 32P-dATP.
Northern blot analysis
Northern blot analysis was used to detect mRNAs for b2-M and the b2-M receptor, as previously explained (43). In brief, poly(A)+ RNA was subjected to 0.8% agarose gel electrophoresis under denaturing conditions, and RNA was vacuum transferred to GeneScreen Plus membranes. Each membrane was prehybridized and then hybridized in buffer with 32P-b2-M (5.2 x 107 cpm) or 32P-b2-M receptor (3.7 x 107 cpm) cDNA probes at 60 C for 20 h, subjected to stringent washing conditions (60 C), and then exposed to a GS-250 Imaging Screen-BI and the GS-250 Molecular Imager (Bio-Rad). Size of each band of b2-M or b2-M receptor mRNAs was determined based on distance migrated in each gel (Rf) for each RNA standard.
Preparation of spent media and follicular fluid for immunoblot analysis of 2-M
To identify 2-M in spent media, granulosa cells were obtained from two dominant follicles and cultured (2 x 106 cells/ml media) in quadruplicate in serum-free Ham’s F12 media supplemented with 4 µM 19-hydroxyandrostenedione for 18 h. After culture, 900 µl of media were removed from each well and centrifuged at 3000 x g for 2 min at room temperature to remove cell debris. To maximize amounts of 2-M for immunoblot analysis, supernatants were pooled within follicles, and a 3-ml sample was desalted with use of an Econo-Pac 10DG column. Protein concentration was determined spectrophotometrically, and samples were stored at –20 C until immunoblot analysis.
To identify 2-M in follicular fluid, bFF (90 µl) was aspirated from each follicle and immediately treated with a protease inhibitor cocktail (10 µl of a 10 x solution in DPBS) to minimize transformation of 2-M during sample processing, and samples were stored at –20 C.
Preparation of lysates for immunoblot analysis of 2-M, 2-M receptor and aromatase
To prepare cell lysates for immunoblot analysis of 2-M and the 2-M receptor, granulosa cells from dominant and subordinate follicles were gently scraped into 100 µl DPBS, immediately frozen in liquid nitrogen, and stored at –80 C. After thawing, cells were exposed to lysing buffer (DPBS, 0.1% Triton X-100, protease inhibitor cocktail) and triturated through a 22-gauge needle attached to a syringe. Granulosa cell lysates were centrifuged at 1000 x g for 5 min at 4 C, and supernatants were stored neat at –20 C or desalted with use of a Microcon centrifugal device and stored at –20 C. To prepare cell lysates for immunoblot analysis of the aromatase enzyme, granulosa cells were cultured under serum-free conditions. At the end of each culture period, media were removed, and granulosa cells remaining in each well were washed twice with DPBS, centrifuged (400 x g, 5 min) at room temperature, and cell pellets were lysed and stored at –20 C. Protein concentrations in lysates were determined with use of DC protein assay kit.
Immunoblot analysis
Immunoblot analysis was used to detect b2-M in bFF, cell lysates, or spent media after culture of granulosa cells and b2-M receptor or aromatase in cell lysates. Details of immunoblot procedures are similar to those previously published (44). In brief, all proteins were subjected to native 5% PAGE, or 5 or 10% SDS-PAGE under nonreducing or reducing conditions with use of the Bio-Rad Minigel apparatus. After electrophoresis, proteins were transferred to Immobilon-P membranes, which were blocked overnight in 1% Blotto, and then incubated (b2-M blots = 2 h; b2-M receptor and aromatase blots = 4 h) with a 1:1000 dilution of anti-b2-M, anti-h2-M receptor, or antiaromatase antibodies in 5% Blotto. After incubations, membranes were washed and incubated in second antibodies (b2-M blots = 1:2000 dilution donkey antirabbit IgG-horseradish peroxidase; b2-M receptor and aromatase blots = 1:1000 dilution sheep antimouse IgG-horseradish peroxidase). All membranes were washed and subjected to enhanced chemiluminescence Western blotting detection reagents for 1 min per manufacturer’s instructions. Membranes were then exposed (b2-M = 1–10 sec, b2-M receptor = 19 h, aromatase = 2–4 h) to film. In some studies, intensity of bands was analyzed with use of the Bio-Rad model GS-670 imaging densitometer and the Molecular Analyst software program, and results were arbitrarily expressed as units. After silver staining gels, molecular weights were estimated with use of high-molecular-weight calibration kit and broad-range protein standards.
To validate use of immunoblots to estimate alterations in relative amounts of total b2-M (native + transformed) in bFF, or b2-M receptor or aromatase in granulosa cell lysates, different amounts of bFF (10, 20, 30, 40 µg), b2-M (0.1, 0.5, 0.75, 1 µg), or granulosa cell lysates (5, 10, 20 µg) were subjected to immunoblot analysis. Intensity of 360-kDa b2-M band in bFF, or the 500-kDa b2-M receptor or the 51-kDa aromatase bands in granulosa cell lysates, was linearly (P < 0.01) associated with amounts of protein subjected to immunoblot analysis (data not shown). In addition, incubation of anti-b2-M with excess b2-M before immunoblot analysis eliminated detection of the 360-kDa b2-M band, thus demonstrating the specificity of anti-b2-M polyclonal antiserum (data not shown). Specificity of monoclonal antibodies was not tested.
Serum-free culture of granulosa cells
Unless specified otherwise, all in vitro studies use the short-term (
18 h) serum-free culture system developed and validated in our laboratory for bovine granulosa cells isolated from individual dominant or subordinate follicles (33, 38). In brief, each treatment for cells from each follicle is in triplicate in serum-free Ham’s F12 media without hormonal or growth factor additives other than the addition of androgen substrate and b2-M, h2-M, BSA (control) or bIgG (control). Granulosa cells (1 x 105 cells/200 µl media per well, unless specified otherwise) were cultured in 96- or 24-well plates containing Ham’s F12 medium supplemented with 1 µM 19-hydroxyandrostenedione and the various treatments previously equilibrated at 37 C. Granulosa cells were incubated at 37 C in a humidified atmosphere (5% CO2 and 95% air) for 0–18 h. After culture, spent medium was carefully removed, centrifuged, and stored at –20 C until determination of estradiol concentration or immunoblot analysis.
RIA
Concentrations of estradiol and progesterone in nonextracted bFF and concentration of estradiol in media were determined by RIA using commercially available kits, as previously validated (43). Sensitivity of the estradiol assay was 0.5 pg/ml, and intra- and interassay coefficients of variation (CVs) were 6 and 7%, respectively. Sensitivity of the progesterone assay was 0.1 ng/ml, and intra- and interassay CVs were 5 and 9%, respectively.
Statistical analysis
Number of follicles and cows used in each cell culture experiment is explained in detail in Results and figure legends. In general, each cell culture experiment was replicated at least twice using granulosa cells primarily from individual follicles obtained from more than 100 cows at a local abattoir. Within each experiment, each treatment was replicated two to four times. Overall treatment effects were determined using the general linear model procedure of SAS (45). Concentrations of estradiol in media were log transformed to satisfy assumptions of normally distributed errors before statistical analyses, but actual values are reported. Overall treatment effects of P < 0.05 were considered significant. If treatment effects were significant, Bonferroni t test was used to determine whether individual means differed (P < 0.05). Linear regression analysis was performed to determine the relationship between intrafollicular alterations in estradiol, 2-M, and the 2-M receptor.
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Results |
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2-M and 2-M receptors2-M and a 15-kb mRNA for the b2-M receptor in granulosa cells of dominant and subordinate follicles (Fig. 1A
). Immunoblot analysis of b2-M, spent media, granulosa cell lysates, and bFF showed that a single predominant 360-kDa band was detected by the anti-b2-M antiserum in each sample (Fig. 1B), whereas a predominant 500-kDa band, which corresponds to the -chain of the 2-M receptor (8), was detected by anti-h2-M receptor antibody in granulosa cell lysates (Fig. 1C).
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2-M used to treat granulosa cells in vitro2-M and h2-M were predominantly native, rather than proteinase-activated or oxidized, aliquots of b2-M, and for comparison bFF, were each treated with or without an equal volume of 10-fold molar excess of bovine trypsin. Similar to other reports (2, 3, 46), trypsin treatment caused 2-M in the commercial b2-M preparation (2-M, +T; Fig. 2) and bFF (bFF, +T; Fig. 2) to migrate faster on native gels, compared with untreated samples (–T). Because our immunoblot detected no bands in any gel lanes of b2-M or bFF below the trypsin-treated b2-M or bFF bands, it is highly unlikely that the commercial preparation of b2-M or the 2-M in bFF had oxidized significantly during storage. However, we cannot rule out the possibility that the anti-b2-M antiserum does not detect oxidized 2-M, or the amounts of oxidized 2-M in the commercial preparation of b2-M or bFF are below the limit of detection of our assay. Results similar to those just described were also achieved with a commercial preparation of h2-M (data not shown). Silver stain analysis, however, revealed that the h2-M preparation was more highly purified than the b2-M preparation, probably because BSA was added to the commercial bovine preparation (data not shown). Based on immunoblot analysis, we concluded that bFF and the commercial preparations of b2-M or h2-M contained significant amounts of the native form of 2-M, which have the capacity to inhibit proteinase activity and bind growth factors. Therefore, both commercial preparations were used in in vitro studies.
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2-M treatments on capacity of granulosa cells to produce estradiol2-M, which is in relatively high concentrations in bFF (18) (Fig. 2
), altered the capacity of granulosa cells to produce estradiol in vitro. To address this question, granulosa cells were isolated from dominant and subordinate follicles, and cells from each follicle were treated with 1 mg of bIgG or b2-M during culture. Bovine 2-M increased (P < 0.01) capacity of granulosa cells isolated from the different size follicles to produce estradiol 20- to 40-fold, compared with controls (Fig. 3).
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2-M was examined in the previous study, the next study tested whether the effect of 2-M on capacity of granulosa cells to produce estradiol was dose responsive. For this study, granulosa cells were isolated from first-wave dominant follicles, and cells from each follicle were treated with bIgG, b2-M, or h2-M during culture. Doses of b2-M spanned the physiological ranges for the average concentrations (0.6 to 1.2 mg) of b2-M per milliliter of bFF (17, 18). Higher doses of b2-M were tested because h2-M was more highly purified, as explained earlier. Both b2-M and h2-M increased (P < 0.01) capacity of granulosa cells to produce estradiol in a linear dose-response fashion to maximal levels that were 10- to 12-fold greater (P < 0.01) than controls (Fig. 4). The more highly purified h2-M was an approximately 18-fold more potent (P < 0.01) enhancer of estradiol production by granulosa cells than b2-M.
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2-M, and 2-M receptor in dominant and subordinate follicles2-M, and the 2-M receptor were positively associated, bFF, and granulosa cells were isolated from first-wave dominant and subordinate follicles for each pair of ovaries from six cows. Concentration of estradiol was determined in bFF, whereas amount of total 2-M or amount of the 2-M receptor was estimated in bFF and granulosa cell lysates, respectively. Data were aligned relative to estradiol concentrations in bFF and subjected to regression analysis. The results demonstrated that although amounts of total 2-M in bFF were unchanged, amounts of 2-M receptor in granulosa cells were inversely (P < 0.05, r = –0.53) associated with intrafollicular concentrations of estradiol, which ranged from 0.03 to 130 ng/ml in dominant or subordinate follicles (Fig. 5).
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2-M on capacity of granulosa cells from individual dominant nonovulatory or dominant ovulatory follicles to produce estradiol2-M is altered during dominant follicle development, granulosa cells were isolated from estrogen-active or estrogen-inactive dominant follicles. Granulosa cells from estrogen-active follicles have a relatively high basal capacity to produce estradiol, and FSH stimulates these cells to produce estradiol and progesterone in a dose-responsive fashion (38). In contrast, granulosa cells from estrogen-inactive follicles have a low capacity to produce estradiol, and FSH stimulates these cells to produce progesterone but not estradiol in a dose-response fashion (38). Granulosa cells from both of these diverse stages of dominant follicle differentiation were cultured and treated with 1 mg of bIgG or b2-M. The results showed that granulosa cells isolated from estrogen-active dominant follicles basally produced 5-fold greater (P < 0.01) amounts of estradiol during culture, compared with cells from estrogen-inactive dominant follicles (Fig. 6A). Nevertheless, b2-M increased (P < 0.01) capacity of granulosa cells from both estrogen-active and estrogen-inactive follicles to produce estradiol 10- and 13-fold, respectively (Fig. 6A).
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2-M enhanced estradiol production by granulosa cells isolated from ovulatory and nonovulatory dominant follicles of conscious cows. The results showed that b2-M increased (P < 0.05) capacity of granulosa cells from dominant ovulatory and nonovulatory follicles 2.5- and 2.2-fold, compared with untreated controls (Fig. 6B).
Specificity of the 2-M-induced increase in estradiol production
To examine the specificity of 2-M action, granulosa cells were isolated from at least two dominant follicles and cells from each follicle were cultured with different doses of b2-M (0, 0.5, or 1 mg), h2-M (0, 10, or 20 µg), and/or the following diverse factors that could alter capacity of granulosa cells to produce estradiol: hormones known to stimulate estradiol production by bovine granulosa cells (23, 38): bFSH or ovine FSH (0.1, 1, 5, and 10 ng) or IGF-I (1, 10, 100, and 400 ng); hormones, growth factors, or binding proteins that bind 2-M (1, 47, 48): bLH (0.1, 0.5, 1, and 10 ng), recombinant human (rh)TGF (0.1, 1, 10, and 100 ng), bovine insulin (1, 10, and 100 ng), osteogenic growth factor (0.001, 0.01, 0.1, 1, 10, 100, and 1000 ng), TGFß1 (0.01, 0.1, 1, and 10 ng), EGF (1, 10, and 100 ng), and follistatin (1 and 5 µg); binding protein that inhibits steroidogenesis by granulosa cells (49) and may bind 2-M: rhIGF-binding protein-4 (10 and 100 ng); various doses (2.5, 5, 25, and 50 pg; 50 pg of extract is equivalent to the amount of extract in 1 mg BSA or b2-M) of a 25-mg extract (50) of b2-M, which may contain TGFß and other similar growth factors (50), or BSA (control); antibodies against growth factors produced by thecal and granulosa cells (51, 52) that modulate FSH-induced estradiol production (28, 53): antihuman TGF (60 µg) or anti-TGFß (10, 50, and 100 ng); non-2-M factors that bind the 2-M receptor (1): human lactoferrin (0.01, 0.1, and 1 µM) or low-density-lipoprotein (0.01, 0.1, and 1 µM); cell attachment or matrix factors: bovine vitronectin (500 ng), BSA (1, 10, 60, 100, 500, and 1000 µg), bIgG (10, 100, 250, 500, and 1000 µg), charcoal-extracted serum from an ovariectomized cow [0.5, 2.5, and 5% (vol/vol)], or charcoal-extracted bFF [0.5, 2.5, and 5% (vol/vol)]; or different cell culture media: Ham’s F12 with or without phenol red, DMEM, or RPMI 1640. Also, high doses of b2-M used to treat granulosa cells could interfere with RIA of estradiol in spent media. Consequently, we tested whether a 1-mg dose of b2-M, which was equivalent to the highest dose used to treat granulosa cells, interfered with the estradiol RIA.
The results of these studies demonstrated that other than b2-M or h2-M, only FSH increased estradiol production by granulosa cells (data not shown). Specifically, bFSH increased capacity of granulosa cells to produce estradiol in a linear dose response fashion (data not shown); and, at the maximal FSH dose tested (10 ng), estradiol concentrations were 2-fold greater (P < 0.01) in FSH-treated cells, compared with untreated controls (data not shown). In the same study, however, treatment of granulosa cells with the maximal dose of b2-M (1 mg) increased estradiol production approximately 12-fold, compared with untreated controls (data not shown). In addition, none of the other variety of factors tested mimicked or altered 2-M-induced increase in estradiol production by granulosa cells, and 2-M did not cross-react in the estradiol RIA (data not shown).
Mechanism of 2-M action
Whether the b2-M-induced increase in estradiol production was caused by an increase in viability or number of granulosa cells was examined as follows. Granulosa cells isolated from two dominant follicles were pooled and cultured with or without (n = 12 replicates) 0.5 mg b2-M. At the end of culture, granulosa cells were removed from each culture well via trituration and subjected to the Trypan blue exclusion dye to assess viability or to a Coulter counter to assess cell number. Treatment of granulosa cells with b2-M did not alter (P > 0.10) cell viability (mean ± SEM; 64.4 ± 2.6% vs. 63.9 ± 2.2%; n = 6 replicates/treatment) or cell number (1.17 ± 0.06 x 105 vs. 1.27 ± 0.03 x 105 cells; n = 6 replicates/treatment).
Oxidants produced during cell culture could conformationally transform b2-M and enable it to bind its receptor (54) and thus modify the b2-M-induced increase in estradiol. To examine the possible effects of oxidants generated during cell culture on 2-M action, granulosa cells were isolated from two dominant follicles and the effect of b2-M (0, 0.5, 1 mg) treatments in the absence or presence of different doses (0, 0.5, 1, 2, and 4 mM) of the potent antioxidant glutathione on estradiol production by granulosa cells from each follicle was tested. The results showed that glutathione did not alter the b2-M-induced increase in estradiol production (data not shown).
Matrix metalloproteinase-9 is produced by bovine granulosa cells during short-term serum-free culture (Ireland, J. J., F. Jimenez-Krassel, and G. W. Smith, unpublished results) and granulosa cells from numerous species produce proteinases other than metalloproteinases (55), such as plasminogen activators (56, 57). Consequently, proteinases produced by live or dead granulosa cells during serum-free culture may not only inhibit in vitro capacity of granulosa cells to produce estradiol by destruction of growth factors or other unknown factors involved in regulation of aromatase bioactivity but could also modify proteins or peptides used to treat granulosa cells in vitro. To test the possibility that alterations in capacity of granulosa cells to produce estradiol in response to 2-M may be explained by the universal capacity of 2-M to inhibit proteinases detrimental to steroidogenesis, granulosa cells were isolated from dominant nonovulatory follicles. The effects of TIMP-1 (recombinant ovine TIMP-1: 10, 20, 100, 200 µg) or the protease inhibitor cocktail (inhibits a broad spectrum of serine, cysteine, and metalloproteinases as well as calpains) were then compared with the effects of 2-M on estradiol production by granulosa cells from each follicle. Doses of recombinant ovine TIMP-1 tested spanned the physiological ranges for TIMP-1 (0.1 µM) (58), whereas doses of protease inhibitor cocktail were based on the maximal doses suggested in the manufacturer’s instructions. The results demonstrated that recombinant ovine TIMP-1 had no effect on estradiol production (data not shown). In contrast, the protease inhibitor cocktail decreased (P < 0.01) basal and the 2-M-induced increase in estradiol production in a dose-response fashion (Fig. 7).
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2-M-induced increase in estradiol production by granulosa cells, we tested whether cAMP, or isobutyl-methylxanthine (IBMX; inhibits phosphodiesterases that degrade cAMP) mimicked 2-M action. In this study, granulosa cells were isolated from nonovulatory dominant follicles, and cells from each follicle were cultured with different doses of b2-M (0.01, 0.1, 0.5, and 1 mg) or dibutyryl cAMP (0.01, 0.1, and 1 mM) and/or IBMX (0.1, 0.5, 1, and 5 mM). Results show that neither dibutyryl cAMP or IBMX altered estradiol production (data not shown).
Whether the b2-M-induced increase in estradiol was explained by an increase in amount of aromatase enzyme in granulosa cells was examined. In this study, granulosa cells were isolated from dominant nonovulatory follicles and treated with BSA or h2-M for 0, 3, or 6 h. At the end of each culture period, media were removed and subjected to estradiol RIA, whereas granulosa cells were lysed, and lysates (5 µg) were subjected to immunoblot analysis to detect the aromatase enzyme. Intensity of the 51-kDa band detected in each sample (Fig. 8A) was determined. Despite the marked increase (P < 0.01) in estradiol production by granulosa cells treated with h2-M, compared with controls (Fig. 8C), the amount of the aromatase enzyme decreased (P < 0.01) similarly (P > 0.10) for both BSA and h2-M treatments (Fig. 8B).
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2-M in regulation of estradiol production by granulosa cells. Surprisingly, however, amounts of the 2-M receptor in granulosa cells were strongly inversely related to intrafollicular concentrations of estradiol during development of dominant nonovulatory and subordinate follicles, which are destined for atresia, whereas intrafollicular amounts of total 2-M were unaltered. Whether a similar relationship between intrafollicular concentrations of estradiol and amounts of 2-M and the 2-M receptor in granulosa cells exists during development of dominant ovulatory follicles, which produce much greater amounts of estradiol and are destined to ovulate, compared with dominant nonovulatory follicles, is unknown. Because transformed 2-M binds its receptor and both are rapidly internalized and degraded in lysosomes (9), a relatively low amount of 2-M receptor in estrogen-active dominant follicles, as observed in our study, may result in an enhanced action of native 2-M. Nevertheless, the precise relationship of alterations in intrafollicular estradiol concentrations with changes in amounts of native and transformed 2-M in bFF could not be firmly established in our study, primarily because the denaturing conditions associated with the SDS-PAGE and immunoblot analysis of bFF do not distinguish native 2-M from 2-M transformed by proteinases or oxidation. Whether 2-M and/or the 2-M receptor have a role in enhancement of estradiol production during development of dominant ovulatory follicles when secretion of LH is high or during development of dominant nonovulatory follicles when gonadotropin secretion is low (59) remains to be determined.
Elucidation of the precise intrafollicular relationship of native 2-M, transformed 2-M, and the 2-M receptor with estradiol production is further complicated because of the possible existence of multiple 2-M receptors. Several studies using macrophages indicate that, in addition to the 2-M receptor, which is primarily involved in the rapid uptake and degradation of a diverse variety of proteins (9), an unidentified signaling receptor also exists for transformed 2-M (12, 13, 60, 61). Other studies, however, report that transformed 2-M interacts with the 2-M receptor in neuronal cells to stimulate Ca2+ signaling via N-methyl-D-aspartate receptors (11). Whether granulosa cells, like macrophages, have both an endocytic and a separate signaling receptor, remains to be determined. Nevertheless, recent studies point to a role of the 2-M receptor in pinocytosis of apoptotic cells (62, 63). Interestingly, the highest amounts of 2-M receptor in granulosa cells in our study were observed in follicles with the lowest intrafollicular concentrations of estradiol, which is characteristic of follicles with the greatest numbers of apoptotic granulosa cells (42, 64). The role of 2-M and the 2-M receptor in apoptosis of granulosa cells and follicular atresia, however, is unknown.
The complex biophysical nature of 2-M enables it to simultaneously bind and inhibit the action of most proteinases; bind and modify numerous growth factors potentially involved in growth, differentiation, and function of granulosa cells; and interact with several different receptors. This multiplicity of actions and signaling moieties associated with 2-M complicate elucidation of both its mechanism of action and physiological role in folliculogenesis. For example, the mechanism whereby 2-M stimulates estradiol production by granulosa cells isolated from follicles with a relatively high capacity to produce estradiol (estrogen-active dominant nonovulatory or ovulatory follicles in our study) (59) as well as from early atretic follicles with a low capacity to produce estradiol (estrogen-inactive dominant or subordinate follicles) (59) is unclear. Nevertheless, the 2-M-induced increase in estradiol production was considerably more rapid and robust than that typically observed for bovine granulosa cells after treatments with a variety of growth factors, cytokines, or hormones regardless of culture conditions (23, 28, 29, 30, 31, 32, 65, 66, 67, 68, 69, 70). In addition, the 2-M-induced increase in estradiol production was highly specific and unlikely attributable to growth factors or cytokines bound to commercial preparations of 2-M, factors such as lactoferrin and low-density lipoprotein that bind the 2-M receptor (1), matrix effects during serum-free culture of the relatively high albeit physiological doses of 2-M, or artifacts associated with specific culture media or the estradiol assay.
The highly variable responsiveness of granulosa cells to 2-M during serum-free culture in our studies, as measured by the fold increase in estradiol, may have been the result of differences in number of 2-M receptors in granulosa cells isolated from individual dominant or subordinate follicles at different stages of development; basal production of 2-M by granulosa cells, which may differentially mask effects of 2-M treatments; and production of unknown autocrine or paracrine factors (e.g. growth factors, proteinases) that diminish estradiol production that may, in turn, have had their actions inhibited by 2-M treatments during culture. Also, the 2-M-induced increase in estradiol was probably not mediated by significant alterations in mitosis, apoptosis, or proteolytic destruction of granulosa cells during serum-free culture for several reasons: 1) the majority of the 2-M-induced increase in estradiol production occurred within the first 3 h after treatment, 2) as assessed by Trypan blue exclusion and use of a Coulter counter, 2-M did not alter viability or number of granulosa cells, 3) glutathione, which is a potent antioxidant and antiapoptotic agent (71), did not mimic or enhance the positive effect of b2-M on estradiol production, and 4) treatments of granulosa cells with a variety of proteinase inhibitors did not mimic the 2-M-induced increase in estradiol production by granulosa cells. Although the reason the protease inhibitor cocktail blocked basal and 2-M-enhanced estradiol production is unknown, this observation may point to a role for proteinases in 2-M action. For example, production of proteinases (such as matrix metalloproteinase-9) by granulosa cells during serum-free culture (Ireland, J. J., F. Jimenez-Krassel, and G. W. Smith, unpublished observations), although not detrimental to steroidogenesis, may be necessary to transform native 2-M, thus enabling it to bind to its receptor on granulosa cells and thereby stimulate estradiol production. Alternatively, EDTA, which is a component of the commercial protease inhibitor cocktail, would be expected to chelate ions such as Ca2+ in culture media. Thus, inhibition of Ca2+ efflux could inhibit both basal and the 2-M-induced increase in capacity of granulosa cells to produce estradiol (72, 73).
In support of this possibility, transformed 2-M stimulates Ca2+ uptake by a variety of nonreproductive cells (11, 74). Thus, the EDTA in the inhibitor cocktail may have directly inhibited a fundamental part of the cell signaling mechanism (e.g. Ca2+ efflux) required for 2-M-induced estradiol production. Finally, the 2-M-induced increase in estradiol may primarily involve the well-established ability of native 2-M to bind and modify the actions of growth factors or cytokines (1). For example, 2-M is a serum binding protein for inhibin (75). Inhibin is produced in high quantities during serum-free culture of bovine granulosa cells, and immunoneutralization of inhibin produced basally during culture stimulates a rapid and marked increase in the capacity of granulosa cells to produce estradiol (33). Thus, the 2-M-induced increase in estradiol production could be explained by its ability to bind and neutralize the negative effects of inhibin on estradiol production by granulosa cells. Future studies will, therefore, be necessary to determine whether the mechanism of 2-M-induced estradiol production by granulosa cells is explained by the binding of growth factors such as inhibin to native 2-M or proteinase-induced transformation of native 2-M and the subsequent interaction of transformed 2-M with its receptors.
In our study, the steep rise in estradiol production stimulated by 2-M during the first 6 h of culture occurred despite the concomitant decrease in amounts of the aromatase enzyme in granulosa cells. These observations provide an important clue that the mechanism of action of 2-M involves enhancement in bioactivity, rather than increased synthesis of the aromatase enzyme. Others report that a rapid (minutes) decrease in aromatase activity occurs in neuronal tissues in response to Mg2+, Ca2+, or ATP (76). Although the decrease in aromatase activity in neuronal cells (76) conflicts with the positive effects of 2-M on aromatase activity in our study, it nevertheless supports the possibility that aromatase activity and estradiol production can be rapidly altered, perhaps via nongenomic means. Moreover, the inability of cAMP or cAMP phosphodiesterase inhibitor treatments to mimic the 2-M action in our study supports the possibility that a signaling mode other than cAMP-adenyl cyclase-protein kinase A is involved in regulation of aromatase enzyme activity in granulosa cells isolated from mature antral follicles.
In summary, bovine granulosa cells isolated from dominant or subordinate follicles synthesized and secreted 2-M and contain the 2-M receptor. Treatment of granulosa cells from these follicle types with 2-M markedly enhanced their capacity to produce estradiol. Although intrafollicular amounts of total 2-M (native and transformed) were unaltered, amounts of the 2-M receptor in granulosa cells varied inversely with capacity of dominant and subordinate follicles to produce estradiol. Granulosa cells from both estrogen-active and -inactive dominant or subordinate follicles increased estradiol production in response to 2-M treatments, and 2-M’s mechanism of action involves enhancement of aromatase bioactivity rather than synthesis. Taken together, these results provide strong evidence that 2-M and the 2-M receptor may have previously undescribed autocrine or paracrine roles on granulosa cells potentially important for regulation of estradiol production and dominant follicle growth, differentiation, and atresia.
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Footnotes |
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Abbreviations: b, Bovine; CV, coefficient of variation; DPBS, Dulbecco’s PBS; EGF, epithelial growth factor; FF, follicular fluid; IBMX, isobutyl-methylxanthine; h2-M, human 2-M; 2-M, 2-macroglobulin; rh, recombinant human; TIMP, tissue inhibitor of metalloproteinase.
Received October 20, 2003.
Accepted for publication February 23, 2004.
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References |
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